US6140078A - Salt-inducible promoter derivable from a lactic acid bacterium, and its use in a lactic acid bacterium for production of a desired protein - Google Patents

Salt-inducible promoter derivable from a lactic acid bacterium, and its use in a lactic acid bacterium for production of a desired protein Download PDF

Info

Publication number
US6140078A
US6140078A US09/068,195 US6819598A US6140078A US 6140078 A US6140078 A US 6140078A US 6819598 A US6819598 A US 6819598A US 6140078 A US6140078 A US 6140078A
Authority
US
United States
Prior art keywords
salt
seq
polynucleotide
leu
inducible promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
US09/068,195
Other languages
English (en)
Inventor
Jan W. Sanders
Jan Kok
Gerard Venema
Adrianus M Ledeboer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Unilever Patent Holdings BV
Original Assignee
Unilever Patent Holdings BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Unilever Patent Holdings BV filed Critical Unilever Patent Holdings BV
Assigned to UNILEVER PATENT HOLDINGS reassignment UNILEVER PATENT HOLDINGS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LEDEBOER, ADRIANUS MARINUS, SANDERS, JAN WILLEM, VENEMA, GERARD
Application granted granted Critical
Publication of US6140078A publication Critical patent/US6140078A/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)

Definitions

  • the invention relates to a salt-inducible promoter derivable from a lactic acid bacterium.
  • salt-inducible promoters exist in plants and cyanobacteria; the latter are rather specialised bacteria which can be used for nitrogen-fixation of soil as natural fertilisation and which taxonomically are quite separate from other bacteria.
  • inducible promoter systems are known in Gram-negative bacteria like E. coli and in the Gram-positive bacterium Bacillus subtilis, while recently in WO 95/31563 (Quest International B.V. (A. Nauta c.s.); see ref. 36) an inducible promoter system was described for lactic acid bacteria or their phages, no literature was found relating to salt-inducible promoters active in microorganisms apart from the above mentioned rather specialised cyanobacteria. Although the expression "salt-initiated induction system" was present in said WO 95/31563 (Quest International B.V. (A. Nauta c.s.); ref. 36), no specific salt-initiated induction system was disclosed.
  • the present invention provides for the first time a salt-inducible promoter for lactic acid bacteria and its use in the production of polypeptides by lactic acid bacteria.
  • the invention provides a salt-inducible promoter derivable from a lactic acid bacterium, in particular a salt-inducible promoter the nucleotide sequence of which is present in SEQ. ID. NO: 10 and in FIGS. 6A-H.
  • a salt-inducible promoter active in lactic acid bacteria is first of all that salt is a natural food ingredient and therefore can be used as a food-grade inducer in food fermentation processes. For instance, during the salting stage of cheese curd various processes can be started when the higher salt concentration is used to trigger the formation of various proteins or peptides. Such processes include induced production and secretion of compounds which can contribute to the properties of the final cheese, e.g.
  • a salt-inducible promoter advantageously is a process for the production of any protein or secondary metabolite by a food-grade microorganism, especially a lactic acid bacterium, whereby at the end of the culturing at a high cell density the microorganism is induced by a salt to produce the desired protein or secondary metabolite.
  • salt does only not mean common salt, i.e.
  • sodium chloride but also includes other halides like alkali metal, earth alkali metal and ammonium halides.
  • other halides i.e. bromides and perhaps fluorides, as well as halides with other cations such as substituted ammonium compounds, e.g. tetramethylammonium, or other metallic cations, e.g. Al 3+ , will show the salt-inducing effect.
  • Still another process which can benefit from the presence of a salt-inducible promoter in a microorganism is the production of secondary metabolites, e.g. flavour or taste ingredients, in situ in a fermented product upon the addition of salt, examples of which include dressings and water-containing spreads, as well as sausages and sour dough.
  • the invention also provides a recombinant vector and a transformed lactic acid bacterium each comprising such a salt-inducible promoter.
  • FIGS. 5A-B are identical to FIGS. 5A-B.
  • NS3 Deletion analysis NS3 (see Example 1.3). This shows the schematic drawings of the relevant parts of plasmids pNS3 (10 kb insert), pNS3d (2.4 kb insert), pNS3b (1.0 kb insert), pNS3e (0.5 kb insert), and pNS3f (0.4 kb insert).
  • FIGS. 6A-H are views.
  • FIGS. 7A-B are identical to FIGS. 7A-B.
  • FIGS. 8A-B are identical to FIGS. 8A-B.
  • Prgg rggrL promoter
  • IR inverted repeat
  • PNaCl salt-inducible promoter
  • RNA marker Autoradiogram of a northern blot of total RNA isolated from MG1363 or MGNS3i3 (rggL - ) cells grown in the absence or presence of 0.5 M NaCl. The blot was hybridized with radioactively labeled XbaI-Sau3A fragment encoding the 5' end of orfX. The sizes of an RNA marker are given in the left margin (see Example 1.6).
  • ⁇ -galactosidase activity of NS3 as a function of the NaCl concentration in the culture medium Samples were taken 7 hours after addition of NaCl to a 1:100 diluted culture (see Example 2).
  • FIGS. 13A and 13B are identical to FIGS. 13A and 13B.
  • the translation of both genes is given below the DNA sequence. Translational stops are indicated with asterisk triplets.
  • the transcription start site is shown by the vertical arrow, while the RBS's are in boldface.
  • the XbaI site corresponds to position 1981 in FIG. 4.
  • the EcoRV and ScaI sites were used to make the fusions.
  • Optical density at 600 nm of LL108(pNS378)(squares) and LL108(pNS3PR)(triangles) grown in GM17. NaCl was added to 0.5 M end concentration to part of the culture at A600 0.5 (open symbols); see Examples 3 and 5. N.B. In this specification the indication OD600 (optical density) is used instead of A600 (absorption).
  • FIG. 20 is a diagrammatic representation of FIG. 20.
  • MG1363acmA ⁇ 1 (pVE6007;pNS3AL3), triangles, and
  • NaCl was added to a final concentration of 0.5 M (closed symbols) or 0.1 M (open symbols) to induce the expression of lytPR, acmA, or lacZ, respectively (see Example 5.1).
  • FIG. 21 is a diagrammatic representation of FIG. 21.
  • Cells were induced with 0.025, 0.05, 0.1, 0.25, or 0.5 M of extra NaCl at an optical density of the cultures of 0.5 at 600 nm.
  • M17 containing 0.004 M NaCl was used.
  • PepXP levels were determined two days after induction (see Example 5.1).
  • FIGS. 24A-B are identical to FIGS. 24A-B.
  • FIGS. 25A and 25B are identical to FIGS. 25A and 25B.
  • Strain NS3 was grown in mM17 medium with (A) or without (B) 2% ⁇ -glycerophosphate. ⁇ -galactosidase activity was followed during growth (fat solid lines) either in the presence of 0.3 M NaCl ( ⁇ ) or in the presence of 0.3 M NaCl plus 50 mM glutamic acid ( ⁇ ). Note that the scale of the left Y-axis in A and B is different. The optical density and pH of the cultures are indicated with dotted lines and thin lines, respectively (see Example 6).
  • FIGS. 29A-Q are identical to FIGS. 29A-Q.
  • the invention provides a salt-inducible promoter derivable from a lactic acid bacterium, in isolation from the coding sequence which is normally controlled by said promoter in a wild-type lactic acid bacterium.
  • a salt-inducible promoter was found during the work resulting in the invention and described below in the Examples. This work yielded inter alia a DNA fragment originating from chromosomal DNA of L. lactis MG1363 in which a salt-inducible promoter appeared to be present. The nucleotide sequence of this DNA fragment is given in FIGS. 6A-H and SEQ. ID. NO: 10.
  • Example 1.3-1.3.1 Deletion analysis described in Example 1.3-1.3.1 revealed that salt-induction of the lacZ gene is still possible, when about 1 kb of genomic DNA isolated from L. lactis M1363 is present in front of the lacZ gene (which DNA is present in a HindIII-Sau3A fragment; see polynucleotide 1476-2426 in FIGS. 6D-G).
  • shorter DNA fragments i.e. containing only 540 bp (see the EcoRI-Sau3A fragment containing polynucleotide 1882-2422 in FIGS. 6E-G) or only 440 bp (see the XbaI-Sau3A fragment containing polynucleotide 1982-2422 in FIGS.
  • this part of the rggL gene can be combined with any promoter capable of driving expression of a structural gene in a lactic acid bacterium, but it is preferred that this part of the rggL gene is combined with the real promoter found in the genomic clone, which is the polynculeotide 1926-2000 including an inverted repeat (polynucleotide 1926-1967 of FIG. 6E) instead of a -35 region and including the -10 region (polynucleotide 1987-1992 of FIG. 6E), and with the further part of DNA upstream of the ORFX-encoding gene (see polynucleotide 2001-2068 of FIGS. 6E-F).
  • the genomic clone which is the polynculeotide 1926-2000 including an inverted repeat (polynucleotide 1926-1967 of FIG. 6E) instead of a -35 region and including the -10 region (polynucleotide 1987-1992 of FIG. 6E), and with
  • a preferred essential part comprises the polynucleotide 1482-2068 of SEQ. ID. NO: 10.
  • a salt-inducible promoter comprising the polynucleotide 1-2068 of SEQ. ID. NO: 10 is more preferred, and still more preferred is the use of the full 2.4 kb fragment, thus a salt-inducible promoter, which additionally comprises part of the ORF X gene together forming polynucleotide 1-2426 of SEQ. ID. NO: 10.
  • the invention also provides a modification of a salt-inducible promoter according to the invention or an essential part thereof, which comprises a DNA sequence essentially corresponding to a polynucleotide selected from the group consisting of
  • Essentially corresponding to a polynucleotide is understood as to include genetic variants, such as hybrid sequences containing a salt-inducible promoter or part thereof coupled to other homologous or heterologous DNA sequences including regulatory regions, and sequences containing modifications of the salt-inducible promoter or sequences having mutations, including mutations which still allow hybridization with the complementary strand of the salt-inducible promoter and genetic variants thereof, while still being capable of exerting the promoter function.
  • the rggL gene plays an important role in the effectiveness of the salt-inducible promoter present in the genome of L. lactis MG1363, isolated by the present inventors and used to transform other lactic acid bacteria in order to give them other desirable properties. But it is further believed, that not only the rggL gene indicated by polynucleotide 1095-1925 of SEQ. ID. NO: 10 will be functional in this respect, but also other DNA fragments encoding the same polypeptide, and even other DNA fragments encoding a modification of such rggL polypeptide still having the same or a similar functionality.
  • an aspect of the present invention is a DNA fragment capable of regulating a salt-inducible promoter active in a lactic acid bacterium, which comprises the polynucleotide 1095-1925 of SEQ. ID. NO: 10, or a modification thereof that (a) encodes the same polypeptide as said polynucleotide 1095-1925, or (b) encodes a modification of such polypeptide still having essentially the same regulating capacity.
  • a further aspect of the invention is a recombinant vector comprising a salt-inducible promoter or an essential part thereof as described above, or a DNA fragment capable of regulating a salt-inducible promoter active in a lactic acid bacterium as described above in combination with a DNA fragment selected from the group consisting of a DNA fragment containing the polynucleotide 1926-2000 of SEQ. ID. NO: 10 and modifications thereof still having essentially the same promoting capacity.
  • the invention also provides a transformed lactic acid bacterium comprising a salt-inducible promoter or an essential part thereof as described above, or a DNA fragment capable of regulating a salt-inducible promoter active in a lactic acid bacterium as described above in combination with a DNA fragment selected from the group consisting of a DNA fragment containing the polynucleotide 1926-2000 of SEQ. ID. NO: 10 and modifications thereof still having essentially the same promoting capacity.
  • the promoter, essential parts thereof and other DNA fragments as described above are preferably present in the chromosome of the lactic acid bacterium, but they can also be present as part of a plasmid that can be maintained during growth of the lactic bacterium.
  • the lactic acid bacterium containing a salt-inducible promoter according to the invention either can be the natural host from which the salt-inducible promoter is derivable, or it can be a different lactic acid bacterium. If both the lactic acid bacterium and the salt-inducible promoter applied according to the invention are the same as in the natural situation, the lactic acid bacterium is transformed by incorporating one or more DNA fragments, or the salt-inducible promoter, originating from a lactic acid bacterium, is used in isolation from the coding sequence which is normally controlled by said promoter in a wild-type lactic acid bacterium.
  • the invention provides a process for the production of a desired protein by a transformed lactic acid bacterium, whereby the gene encoding said desired protein or a precursor thereof is expressed under control of an inducible promoter, characterised in that the promoter is a salt-inducible promoter or an essential part thereof according to the invention or a DNA fragment capable of regulating a salt-inducible promoter active in a lactic acid bacterium as described above in combination with a DNA fragment selected from the group consisting of a DNA fragment containing the polynucleotide 1926-2000 of SEQ. ID. NO: 10 and modifications thereof still having essentially the same promoting capacity.
  • the transformed lactic acid bacterium is food-grade due to the use of food-grade DNA sequences and/or removal of non-food-grade DNA sequences.
  • the desired protein is secreted by the lactic acid bacterium due to the presence of a DNA fragment fused to the gene encoding the desired protein and effecting secretion of the desired protein or a precursor thereof.
  • a process according to the invention using a salt-inducible promoter for the expression of a desired gene can be used in a fermentation process, in which the desired protein is a lytic protein causing lysis of the bacterial cells so that the contents of the cells can be released, or in a
  • the desired protein is an enzyme involved in the in situ production of secondary metabolites as flavour or taste ingredients.
  • end products include dressings and water-containing spreads, as well as sausages and sour dough.
  • Still another process which can benefit from the presence of a salt-inducible promoter in a microorganism is a fermentation process, in which the desired protein is a protein having a function in a cheese production process, such as chymosin or a precursor thereof, or an enzyme involved in cheese flavour formation.
  • the invention is exemplified by the following Examples 1-6. preceded by a description of the materials and methods that were used.
  • L. lactis was grown at 30° C. in M17 medium, with 0.5% glucose; solidified M17 medium contained 1.5% agar.
  • Erythromycin (Em) and chloramphenicol (Cm) were used at final concentrations of 5 ⁇ g/ml, spectinomycin (Sp) was used at 100 ⁇ g/ml.
  • 5-Bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside (X-Gal) was used at a final concentration of 0.008%.
  • E. coli was grown in TY broth at 37° C. with vigorous agitation or on TY medium supplemented with 1.5% agar. Ampicillin (Ap) and Em were used at 100 ⁇ g/ml, Sp at 50 ⁇ g/ml.
  • DNA and RNA techniques were performed essentially as described by Sambrook c.s. (see ref. 16). DNA was introduced by electrotransformation in E. coli (see ref. 19; E. R. Zabarovsky & G. Winberg; 1990) and in L. lactis (see ref. 15; H. Holo & I. F. Nes; 1989). DNA sequencing was done on double-stranded plasmid DNA by the dideoxy chain-termination method (see ref. 4; F. Sanger c.s.; 1977) and the T7 sequencing kit (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) according to the manufacturer's instructions.
  • oligonucleotides were synthesized with an Applied Biosystems 392A DNA synthesizer (Applied Biosystems Inc. Foster City, Calif.). DNA sequences were analyzed with the PC/Gene sequence analysis program (IntelliGenetics Inc., Geneva, Switzerland). Protein homology searches against the Genbank were carried out using the FASTA program (see ref. 12; W. R. Pearson & D. J. Lipman; 1988). Protein sequence alignments were carried out with the PALIGN program of PC/Gene using the structure genetic matrix or with the CLUSTAL program, both with standard settings.
  • Cell extracts were prepared from exponentially growing cultures. ⁇ -Galactosidase activity was determined as described by Miller (see ref. 2; J. H. Miller; 1972). Protein concentrations in the cell extracts were determined by the method of Bradford (see ref. 3; M. M. Bradford; 1976) with bovine serum albumin as a standard.
  • helper strains were constructed containing the repA gene of pWV01, i.e. L. lactis LL108 and L. lactis LL302 (see 1.1.2 below) and E. coli EC1000 (see 1.1.1 below). This work is based on the technology described in EP-A1-0 487 159 (see ref. 26; Unilever N.V./PLC (C. J. Leenhouts c.s.); 1992). Subsequently several plasmids were constructed:
  • These plasmids carry the ORI + of pWV01 but lack the repA gene for the replication initiation protein. However, they can be replicated in the helper strains containing the repA gene of pWV01.
  • the promoter screening vector pORI13 was constructed (FIG. 1). To allow only transcriptional fusions one stop codon in each reading frame was present immediately upstream of the E. coli lacZ gene.
  • the lacZ gene is preceded by lactococcal translation signals derived from ORF32 (see ref. 24; M. van de Guchte c.s.; 1991 and ref. 10; J. M. B. M. van der Vossen c.s.; 1987). Since plasmid pORI13 carries the ORI + of pWV01 but lacks the repA gene for the replication initiation protein, it can be used for Campbell-type integrations. Random Sau3A fragments obtained from total chromosomal DNA of L.
  • the repA gene from pWV01 was introduced onto the chromosome of E. coli MC1000 as described for JM101 by Law c.s. (see ref. 37; J. Law c.s.; 1995).
  • pKVB2 (see ref. 37 and ref. 11; J. A. K. W. Kiel c.s.; 1987) is a TC r Km r plasmid of 11.7 kb containing the origin of replication of pBR322. It carries the E. coli chromosomal glgB gene in which an internal 1.2 kb BamHI fragment was replaced by the Km r gene from the Streptococcus faecalis plasmid pJH1.
  • the resultant plasmids pEC1 and pEC2 differ solely in the orientation of repA (see ref. 37; J. Law c.s.; 1995).
  • Plasmids pEC1 and pEC2 were used to transform E. coli JM101. Before plating on selective media the transformation mixtures were transferred for 30 generations in the absence of antibiotic and then plated on Km-containing plates. Colonies were tested for glycogen production. Non-glycogen producing colonies were transferred onto plates containing Km and Tc and onto plates containing Km alone. Km r TC s colonies were found and were plasmid-free and contained repA integrated at the specific site on the chromosome. Confirmation of the RepA + nature of one of the strains (E. coli EC1000) was obtained by the successful transformation of this strain with an ORI + RepA - plasmid. This work will also be described in a publication by Leenhouts c.s. accepted by Mol. Gen. Genet. for publication in 1996 (see ref. 39; K. Leenhouts c.s.; 1996).
  • Plasmid pKL15A is a derivative of the pBR322-based Campbell-type integration plasmid pHV60 (see ref. 14; K. J. Leenhouts; 1989) in which the repA gene from plasmid pUC23rep3 (see ref. 22; K. J. Leenhouts; September 1991) was inserted. Integration of pKL15A into the chromosome of L. lactis MG1363 by selection for chloramphenicol resistance resulted in L. lactis strain LL108 carrying approximately 15 tandem copies of the integration plasmid, as has been described earlier for similar plasmids (see ref. 14; K. J. Leenhouts; 1989).
  • the replacement-type integration vector pUK was obtained by cloning the repA fragment of pUC23rep3 in the multiple cloning site of pUK29.
  • the latter plasmid is a derivative of pUK21 (see ref. 23; J. Vieira & J. Messing; 1991) and carries the Em resistance gene of pUC19E (see ref. 17; K. J. Leenhouts; 1990) in the XhoI site, the 3'-end of the L. lactis pepXP gene (see ref. 20; B.
  • Plasmid pUK30 was used in a two-step gene-replacement strategy (see ref. 21; K. J. Leenhouts c.s.; August 1991) for transforming L. lactis MG1363 yielding L. lactis strain LL302.
  • This strain contained one copy of pWV01 repA inserted in the pepXP gene.
  • Both strain LL302 and strain LL108 allow the replication of pWV01-based vectors which lack repA. This work will also be described in a publication by Leenhouts c.s. submitted for publication in 1996 (see ref. 40).
  • the Tc r of PLS1 was introduced in the SmaI site of pMTL25 (see ref. 13; S. P. Chambers c.s.; 1988) to produce pTC2.
  • This Tc resistance gene was isolated from plasmid pTC2 as a 1.6 kb BamHI fragment and plasmid pUK21 (see above) was digested with XhoI. Prior to ligation of the two fragments, blunt ends were generated by Klenow enzyme treatment. The ligation resulted in pUK24, in which the Tc r gene is flanked by two XhoI restriction sites.
  • the lacZ gene of pMG60 was cloned in pKL10 (see ref. 17; K. J. Leenhouts; 1990) in two steps.
  • the 1250 bp SspI fragment ex pMG60 containing the 5'-end of lacZ gene and the lactococcal RBS of ORF32, was ligated with the Klenow treated XbaI site of pKL10.
  • the resulting construct, PLS10 was restricted with HindIII, the HindIII sticky ends were made blunt and then the fragment was treated with ClaI.
  • the resulting fragment was ligated with a ClaI-XmnI fragment of pMG60 containing the additional part of the lacZ gene, including a transcriptional terminator, resulting in pLS11.
  • the multiple cloning site (mcs) of pBSK+ had to be inserted upstream of the RBS preceding the lacZ gene.
  • mcs multiple cloning site
  • the mcs of pBSK+ it was marked by cloning a BamHI fragment from pUC7K, coding for a Km r gene, into the BamHI site of the mcs.
  • the mcs was excised from this plasmid using NotI and XhoI. The sticky ends were made blunt-ended and the fragment was ligated into the BamHI site (also made blunt-ended) of pLS11 in front of the lacZ gene.
  • the Km r gene was deleted using BamHI followed by self ligation. This resulted in the integration expression vector pLS12 (see FIG. 2).
  • the erythromycin resistance gene of pLS12 was replaced by that from pORI28, including a multiple cloning site by isolating a StuI-XbaI fragment from pORI28. This fragment was ligated into pLS12 digested with EcoRI, made blunt-ended with Klenow enzyme and subsequently digested with XbaI. The resulting construct was designated pLS13 (see FIG. 3).
  • the E. coli lacZ gene fused to lactococcal translation signals, as present on pMG60 was used.
  • a 2.5 kb ClaI-XmnI fragment of pMG60 was ligated in XbaI-ClaI linearized pORI28 of which the XbaI site was made blunt using Klenow.
  • the ligation mixture was digested with EcoRI to prevent replication of pMG60 and used to transform the RepA + lactococcal helper strain LL108.
  • the resulting construct, pLS28 was cut with BglII and BssHII.
  • the 5' end of the lacZ gene was liberated from pLS13 using the same restriction endonucleases. This 1.6 kb fragment was ligated to pLS28 and the ligation mixture was used to transform the RepA + L. lactis helper strain LL302.
  • the resulting plasmid was designated pORI13 (see FIG. 4).
  • Total chromosomal DNA of L. lactis MG1363 was partially digested with Sau3A to obtain fragments ranging in size from 1 to 10 kb which were ligated to BamHI and alkaline phosphatase treated pORI13. The chromosomal fragments were ligated in the linearized pORI13. This ligation mixture was used to transform the RepA + E. coli helper strain EC1000 (see 1.1.1 above). Transformants were collected from agar plates by pouring 2 ml of TY broth on each plate, and their plasmid DNA was isolated.
  • the plasmid mixture obtained in the previous step was used to transform L. lactis MG1363(pVE6007). After electroporation, cells were suspended in recovery medium of 30° C. (see ref. 15; H. Holo & I. F. Nes; 1989). After 1.5 hour 5 ⁇ g/ml erythromycin was added and incubation at 30° C. was prolonged for 0.5 hour. Cells were shifted to 37° C. for 2 hours and then plated on sucrose (0.5 M) GM17 agar containing X-gal, erythromycin and 0.3 M NaCl. Incubation was at 37° C. for 24 hours and subsequently at 30° C. The recovery of pORI13 derivatives from the chromosome of selected integrants was done as described by Law c.s. (see ref. 37; J. Law c.s.; 1995).
  • Colonies expressing ⁇ -galactosidase in the presence of NaCl were transferred to GM17 agar plates with or without 0.5 M NaCl. Of the 195 selected blue colonies (see 1.1 above) 80 were white on the NaCl-free plates, indicating the absence of ⁇ -galactosidase expression. The intensity of the blue colour in the presence of NaCl was similar for all 80 clones.
  • the integrated pORI13 derivatives in 5 of these clones, called NS1-NS5 were rescued and appeared to be identical at the restriction enzyme level. All five plasmids expressed the salt-dependent phenotype.
  • pNS3 isolated from clone NS3 was selected for further characterization.
  • pNS3 Restriction enzyme analysis of pNS3 revealed that about 10 kb of chromosomal DNA had been cloned. A number of restriction enzyme sites was used to map the salt-dependent promoter. A PstI deletion (pNS3d) showed that NaCl-dependent lacZ expression is linked to a 2.4 kb fragment directly upstream of the Sau3a site at the original fusion point (see FIG. 5 and position 2423 of FIG. 6G).
  • pNS3 pNS3 was digested with HindIII yielding a fragment carrying lacZ and 1.0 kb of upstream lactococcal DNA. This fragment was ligated to a HindIII fragment of pORI13 carrying the Em resistance marker and the plus origin of replication of pWV01.
  • the resulting construct, pNS3b was isolated from E. coli EC1000.
  • pNS3d was made by self-ligation of PstI digested pNS3 and carries 2.4 kb of chromosomal DNA fused to lacZ. The proper construct was obtained in L.
  • Plasmid pNS3e was obtained by using the self-ligation mixture of an EcoRI digest of pNS3 to transform E. coli EC1000 and carries an about 540-bp chromosomal DNA fragment upstream of lacZ.
  • pNS3f was constructed by ligating the XbaI fragment from pNS3III (see 1.4.1 below) to the XbaI site of pORI13 and transformation of E. coli EC1000.
  • pNS3f carries an about 440 bp chromosomal DNA fragment upstream of lacZ (see FIG. 5B).
  • OrfX is preceded by a ribosome binding site (RBS).
  • RBS ribosome binding site
  • an ORF with a weak RBS could encode a protein of 276 amino acid residues (see SEQ. ID. NO: 10 and 11) with a calculated molecular weight of 32990. It shows homology to the Streptococcus gordonii rgg gene product (see SEQ. ID. NO: 12), which regulates expression of the glucosyl transferase gene (see ref. 27; M. C. Sulavik c.s.; 1992) and that of a partially sequenced ORF downstream of the L. lactis pip gene (see FIGS. 7A-B, SEQ. ID. NO: 13 and ref. 30; B. L.
  • FIG. 9 The genomic organization of the NS3 locus is given in FIG. 9, which shows that two rare restriction enzyme sites are present on the sequenced fragment, one for NotI (nucleotides 306-313, FIG. 6A) and one for ApaI (nucleotides 2382-2387, FIG. 6F), separated by only about 2070 bp. From a comparison with the genetic map of L. lactis MG1363 (see ref. 32; P. Le Bourgeois c.s.; 1995) it is clear that the NS3 locus is positioned between the ldh gene and the leu-ilv gene cluster.
  • the 2.5 kb region upstream of the lacZ-fusion point in the chromosome of L. lactis NS3 was recovered into several subclones and sequenced.
  • a 401 bp EcoRI-HindIII fragment from pNS3b was cloned in pUC19 and designated pNS3bI.
  • 784 bp and 470 bp XbaI fragments from pNS3 were cloned in the XbaI site of pUC18, resulting in pNS3II and pNS3III, respectively.
  • a 604 bp HindIII fragment from pNS3 was cloned in pUC18 and the plasmid was designated pNS3IV and a pUC18 derivative carrying the 743 bp EcoRI-XbaI fragment from pNS3 was called pNS3V.
  • a fragment of 745 bp liberated from pNS3 with HindIII and PstI and cloned in pUC19 resulted in pNS3VI.
  • E. coli NM522 was used as a cloning host for pUC18 or pUC19 constructs.
  • a fragment located at the 3'-end of the chromosomal insert in pNS3 was amplified by inverse-PCR.
  • Northern hybridizations were done at 40° C. in a buffer containing 50% formamide, 7% SDS, 2% blocking reagent (Boehringer, Mannheim, Germany), 5 ⁇ SSC, 50 mM sodium phosphate pH 7, and 0.1% N-lauryl sarcosine.
  • a 470 bp XbaI fragment of pNS3III was used as a probe and labeled with [ ⁇ 32 P-dCTP].
  • a synthetic oligonucleotide (NS3-11) complementary to the mRNA (position +96 to +133 downstream of the transcription start point) was used for primer extension. Twenty five nanogram of primer were added, to 5 ⁇ g of RNA in a reaction mixture containing dCTP, dGTP, dTTP and ⁇ - 35 S-dATP and cDNA was synthesized using AMV reverse transcriptase (Boehringer, Mannheim, Germany). After 10 minutes incubation at 42° C. an excess cold dATP was added and incubation was prolonged for another 10 minutes at 42° C. The product was analyzed on a sequencing gel next to a sequence reaction with the same primer, providing a size marker.
  • the 440 bp XbaI-Sau3A fragment carrying the 5'-end of orfX was used as a probe in Northern hybridization.
  • the size of the NaCl-dependent transcript in MG1363 was estimated to be approximately 3.0 kb.
  • lacZ expression from NS3 is halide-ion-dependent
  • lacZ expression appeared to be independent of the osmolarity of the medium but was strictly linked to the presence of Cl - or I - ⁇ -galactosidase activity increased with increasing NaCl concentrations in the medium (FIG. 12). No induction of ⁇ -galactosidase activity was observed by increasing the growth temperature.
  • the transcription start point upstream of orfX and rggL were amplified as a cassette using primer NS3-7 and NS3-8 and cloned upstream of the holin and lysin genes (lytPR) of the lactococcal temperate bacteriophage r1-t (see ref. 38; D. van Sinderen c.s.; 1996 and ref. 35; Quest International B.V. (A. Nauta c.s.); WO 95/31562).
  • the start codon of orfX was fused
  • FIG. 13A and SEQ. ID. NO: 18 Cultures of the resulting strain, LL108(pNS3PR), were grown in GM17 and induced at an optical density at 600 nm (OD600) of 0.5 by the addition of 0.5 M NaCl.
  • FIG. 14, lane 4 shows that 6 hours after induction, in addition to wild-type autolysin activity, phage lysin activity was present in the induced cells, and absent in the cells grown without NaCl.
  • This fragment was digested with SacI and EcoRV and ligated to pIR1PR (see ref. 35; Quest International B.V. (A. Nauta c.s.); WO 95/31562) linearized with SacI and ScaI.
  • the ligation mixture was used to transform L. lactis LL108 and the resulting plasmid was called pNS3PR (see FIG. 16).
  • the same PCR fragment was cloned upstream of lacZ in pORI13.
  • the PCR fragment was cut with BglII and EcoRV and ligated to BglII-SmaI-digested pORI13.
  • the ligation mixture was used to transform LL108 and the resulting plasmid was labeled pNS378.
  • the cassette containing rggL, the salt-inducible promoter, and the RBS and start codon of orfX was placed upstream of acmA, the gene of the major peptidoglycan hydrolase of Lactococcus lactis (see ref. 34; Quest International B.V. (G. Buist c.s.); WO 95/31561 and ref. 31; G. Buist c.s.; 1995).
  • Two mutations occurred in the fusion region.
  • An A to G transition in the untranslated leader of the transcript see nucleotide 64 in FIGS. 13A+13B and SEQ. ID. NO: 18 and SEQ. ID. NO: 19
  • a deletion of an A residue see nucleotide 99 in FIG.
  • the OD600 of the control strain increased to a higher level in the presence of salt. More AcmA activity was detected in cells induced with NaCl compared to uninduced cells and to control cells expressing ⁇ -galactosidase under control of the same induction cassette (FIG. 14). Clearly, the expression of acmA from pNS3AL3 is induced by NaCl.
  • the acmA gene interrupted by a SacI fragment on pAL10 was used as a basis (see ref. 34; Quest International B.V. (G. Buist c.s.); WO 95/31561).
  • the BGlII sites in pAL10 were deleted by cutting with BglII, filling the overhanging ends with Klenow polymerase, recircularization with T4 ligase and transformation of EC1000.
  • This construct, pAL101 was linearized with BamHI and XbaI and ligated to pORI28, also linearized with BamHI and XbaI.
  • the proper construct, pAL102 was isolated from E. coli EC1000.
  • the ligation mixture was used to transform L. lactis LL302 and pNS3AL3S was recovered.
  • This plasmid was digested with SacI to delete the insert in acmA. After self-ligation the mixture was used to transform LL302.
  • the supernatant of the holin-lysin expressing strain contains a significant amount of cytoplasmic proteins 6 hours after NaCl addition, whereas in the supernatant of the same strain grown without induction only secreted proteins are visible (FIG. 19.A, lane 3).
  • the addition of NaCl to the control strain already causes the release of a small portion of cytoplasmic proteins (FIG. 19.A, lane 2).
  • a much larger quantity of cytoplasmic proteins was observed (FIG.
  • PepXP was chosen as an intracellular marker enzyme.
  • Cell lysis was quantified by measuring PepXP release from cells lacking the chromosomal autolysin gene.
  • MG1363acmA ⁇ 1-(pVE6007) was transformed with either pNS3PR or pNS3AL3.
  • PepXP activity in supernatant samples was determined by following hydrolysis of the synthetic substrate Ala-Pro-p nitroanilide at 405 nm for minutes at 37° C. in a 96-well microtiterplate using a Thermomax microplate reader (Molecular Devices Co., Menlo Park, Calif.).
  • PepXP activity in culture supernatants was followed after induction of either lysin gene with 0.5 M NaCl at a culture OD600 of 0.5.
  • low levels of PepXP were detected in the culture supernatants (FIG. 20).
  • Optimal PepXP levels were obtained 8 hours after induction of cultures of MG1363acmA ⁇ 1(pVE6007)-(pNS3AL3) and 30 hours after induction of MG1363acmA ⁇ 1-(pVE6007)(pNS3PR).
  • the highest level of PepXP activity was present after induction with 0.1 M NaCl (FIG. 21).
  • PepXP activity is stable for at least 40 hours in a cell extract in M17, either with or without NaCl (data not shown).
  • PepXP was extractable with hypoosmotic medium from NS3::acmA carrying cells.
  • the sum of PepXP in the supernatant and PepXP extractable from cells is comparable for NS3::acmA and NS3::lytPR cells induced with 0.25 M NaCl.
  • the amount of PepXP extracted from NS3::acmA cells increased with the amount of NaCl used for induction. Only after induction with 0.5 M NaCl a smaller amount of PepXP was extracted.
  • KCl could replace NaCl as the inducing agent, resulting in only slightly lower levels of released PepXP (data not shown).
  • gad is used to indicate a group of genes involved in glutamate-dependent acid resistance. Therefore, the genes indicated above with rggL and orfX have now been renamed as gadR and gadC, respectively.
  • rggL was discussed in Example 1.4 above.
  • GadB was discussed in Example 1.4 above.
  • GadC is discussed below under the heading RESULTS.
  • Protein homology searches against the Genbank were carried out using the BLAST program (Altschul et al., 1990; ref. 41). Protein sequence alignments were carried out with the PALIGN program of PC/Gene using the structure genetic matrix. Transmembrane segments were predicted using the method of Klein et al. (1985; ref. 45)
  • the region downstream of the lacZ fusion site in L. lactis NS3 was obtained by inverse PCR amplification of a 0.56-kb Sau3A-XbaI fragment from L. lactis MG1363 chromosomal DNA using primers NS3-5 and NS3-11 (see Table 2 above).
  • the PCR product was cloned in pORI19 using Escherichia coli EC101 as a host, resulting in pNS3i4 (see Example 1.4.1 above and FIG. 27).
  • Transformants contained a 4.8-kb plasmid (pNS3i6; see FIG. 27) which is pORI19 containing a 2.6-kb XbaI-EcoRI fragment from the chromosome (FIG. 22).
  • Plasmid pNS3i7 was constructed by cloning a 0.5-kb AsnI fragment (internal to gadb) as a blunt fragment in the SmaI site of pORI19 using E. coli EC101 as a host (see FIG. 27). Single cross-over integration of pNS3i4 and pNS3i7 in the L. lactis MG1363 chromosome resulted in strains MGNS3i4 (gadC) and MGNS3i7 (gadB), respectively.
  • Cell-free extracts were prepared by vigorous shaking of cells in the presence of glass beads (van de Guchte et al.,1991; ref. 24).
  • ⁇ -Galactosidase activity was determined as described by Miller (1972; ref. 2).
  • Protein concentrations were determined by the method of Bradford (1976; ref. 3) with bovine serum albumin as the standard.
  • chloride-dependent promoter P gad
  • P gad chloride-dependent promoter
  • the original Sau3A fusion site in the lacZ integrant L. lactis NS3 is located in an ORF of 503 codons immediately downstream of P gad .
  • This ORF was named gadC, as its deduced amino acid sequence (FIGS. 23A-C) is homologous to GadC from Shigella flexneri (51% identity and 17% similarity, Waterman and Small, 1996; ref. 47) and its E. coli counterpart XasA (Hersh et al., 1996; ref. 43).
  • GadC is homologous to a number of amino acid antiporters, including the lysine-cadaverine antiporter CadB from E. coli (Meng and Bennet, 1992; ref. 46).
  • Lactococcal GadC has a deduced molecular weight of 55369 and a pI of 9.73 and is highly hydrophobic. The hydrophobic residues are clustered in 12 domains (FIGS. 23A-C), whose locations coincide with those of the hydrophobic domains in S. flexneri GadC (as predicted by a number of topology-predicting computer programs). This suggests that GadC is an integral membrane protein. A conserved domain found in glutamate transporting proteins is also present in L. lactis GadC (FIGS. 23A-C, Waterman and Small, 1996; ref. 47); gadC is separated by 19-bp from another ORF of which the deduced protein is homologous to glutamate decarboxylases.
  • gadR is Constitutively Expressed
  • gadR expression of gadR was studied in strain MGNS32 carrying a single copy gadR::lacZ transcriptional fusion (see FIG. 22).
  • ⁇ -galactosidase activity in exponentially growing MGNS32 was 3.0 U/mg, independent of the presence of NaCl.
  • NaCl-dependent expression from P gad is not regulated by variations in the level of transcription of gadR.
  • Another lacZ fusion, located immediately downstream of the 21-bp IR was used to show that transcription of gadR is effectively terminated by this IR. No ⁇ -galactosidase activity could be detected in strain MGNS31 carrying this fusion (see FIG. 22).
  • gadR promoter consists of canonical -35 and -10 hexanucleotides separated by 18 bp (FIGS. 6C and 29C).
  • gadCB is Enhanced at Low pH and by Glutamate
  • ⁇ -galactosidase activity in NS3 was still induced in this modified medium in the presence of 0.3 M NaCl and 2% ⁇ -glycerophosphate buffer but the induction level was fivefold lower as compared to that in standard 1/2M17 (data not shown).
  • the lacZ expression in mM17 in the presence of NaCl was low in the early stages of exponential growth and increased to an optimum at the onset of the stationary phase (FIGS. 25A-B). In the absence of buffer, the culture pH decreased to 4.0 to 4.5 in the stationary growth-phase, while cultures containing buffer reached a lowest pH of 5.5. Expression of lacZ was increased 10-fold in mM17 containing no buffer.
  • Lactic acid was much more deleterious than hydrochloric acid whereas the viability was not affected at pH 6.5.
  • the presence of 1 mM glutamate alone did not affect the viability at pH 3.5.
  • the viability was reduced only 200-fold when lactic acid was used. Under the latter conditions, the viability in MS15 adjusted to pH 3.5 with hydrochloric acid was also enhanced significantly.
  • Lactococcus lactis MG1363 has a glutamate-dependent acid resistance mechanism, that is active in the presence of chloride.
  • L. lactis has two genes, gadC and gads, that encode proteins homologous to a putative glutamate- ⁇ -aminobutyrate antiporter and a glutamate decarboxylase, respectively, from Escherichia coli and Shigella flexneri. These genes are involved in glutamate-dependent acid resistance in E. coli and S. flexneri.
  • gadCB in L. lactis is induced by chloride and is optimal at low pH in the presence of glutamate.
  • L. lactis insertion mutants with a disrupted gadB or that are unable to express both gadB and gadC are more sensitive to low pH than the wild-type when NaCl and glutamate are present, indicating that the lactococcal gadCB operon is involved in glutamate dependent acid resistance, see FIG. 26.
  • FIG. 25A shows that upon induction with 0.3 M NaCl the yield of ⁇ -galactosidase is not more than about 8 Units/mg when the medium is buffered such that the pH will not come below 5.5.
  • FIG. 25B shows that in the absence of a buffer the pH can drop to about 4, while under these conditions the yield of ⁇ -galactosidase is about 80 Units/mg.
  • the salt-inducible promoter is more active at a lower pH and has thus also become pH-inducible in the presence of salt.
  • the activity of the salt- and pH-inducible promoter can be further enhanced in the presence of glutamate/glutamic acid, as is shown in FIGS. 25A and 25B. They show that production of ⁇ -galactosidase is increased from 8 to 15 Units/mg in a buffered medium and from 80 to 225 Units/mg in a non-buffered medium when the pH during fermentation has dropped to about 4.3.
  • Example 6 shows that a desired protein can be produced in a lactic acid bacterium at an improved yield by using a construct in which the gene encoding the desired protein is under control of a salt- and pH-inducible promoter and the medium in which the transformed lactic acid bacterium is cultured is not buffered and preferably contains glutamate/glutamic acid.
  • the desired protein contains a secretion signal sequence to enable secretion of the protein.
  • the transformed lactic acid bacterium also contains at least one gene encoding a lytic protein under control of the salt- and pH-inducible promoter, proteins without a secretion signal sequence can be easily recovered, because the lytic protein will perforate the cell wall so that the contents including the desired protein can be released from the cell.
  • RNAse HII RNAse H
US09/068,195 1996-09-05 1997-08-20 Salt-inducible promoter derivable from a lactic acid bacterium, and its use in a lactic acid bacterium for production of a desired protein Expired - Fee Related US6140078A (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
EP96202444 1996-09-05
EP96202444 1996-09-05
EP97200744 1997-03-13
EP97200744 1997-03-13
PCT/EP1997/004755 WO1998010080A1 (en) 1996-09-05 1997-08-20 Salt-inducible promoter derivable from a lactic acid bacterium, and its use in a lactic acid bacterium for production of a desired protein

Publications (1)

Publication Number Publication Date
US6140078A true US6140078A (en) 2000-10-31

Family

ID=26143129

Family Applications (1)

Application Number Title Priority Date Filing Date
US09/068,195 Expired - Fee Related US6140078A (en) 1996-09-05 1997-08-20 Salt-inducible promoter derivable from a lactic acid bacterium, and its use in a lactic acid bacterium for production of a desired protein

Country Status (7)

Country Link
US (1) US6140078A (de)
EP (1) EP0865496A1 (de)
JP (1) JPH11514534A (de)
AU (1) AU4208797A (de)
CA (1) CA2236770A1 (de)
ID (1) ID18254A (de)
WO (1) WO1998010080A1 (de)

Cited By (95)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030027358A1 (en) * 1997-04-24 2003-02-06 Immunex Corporation Family of immunoregulators designated leukocyte immunoglobulin-like receptors (LIR)
WO2005077116A2 (en) 2004-02-10 2005-08-25 Mosanto Technology, Llc Recombinant dna for gene suppression
US20050281914A1 (en) * 2003-06-20 2005-12-22 Steele James L Methods and compositions involving endopeptidases PepO2 and PepO3
EP1621632A1 (de) 2004-07-31 2006-02-01 Monsanto Technology, LLC Gene und ihre Verwendung zur Pflanzenverbesserung
US20060078564A1 (en) * 2002-05-08 2006-04-13 Immunex Corporation Family of immunoregulators designated leukocyte immunoglobulin-like receptors (LIR)
WO2006073727A2 (en) 2004-12-21 2006-07-13 Monsanto Technology, Llc Recombinant dna constructs and methods for controlling gene expression
WO2007011479A2 (en) 2005-07-19 2007-01-25 Monsanto Technology, Llc Double-stranded rna stabilized in planta
US20070061922A1 (en) * 2005-09-09 2007-03-15 Edgerton Michael D Selective gene expression in plants
WO2007041419A1 (en) 2005-10-03 2007-04-12 Monsanto Technology Llc Transgenic plant seed with increased lysine
WO2007143690A2 (en) 2006-06-06 2007-12-13 Monsanto Technology Llc Methods for weed control
EP1881074A2 (de) 2003-05-05 2008-01-23 Monsanto Technology, LLC Transgene Pflanzen mit erhöhtem Glycin-Betain
US20080040973A1 (en) * 2006-06-23 2008-02-21 Donald Nelson Transgenic crop plants with improved stress tolerance
WO2008048964A2 (en) 2006-10-16 2008-04-24 Monsanto Technology Llc Methods and compositions for improving plant health
US20080104730A1 (en) * 2001-05-22 2008-05-01 Jingrui Wu Yield-improved transgenic plants
WO2008105890A2 (en) 2007-02-26 2008-09-04 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of dmo and uses thereof
WO2008112633A2 (en) 2007-03-09 2008-09-18 Monsanto Technology Llc Method of meristem excision and transformation
WO2008133643A2 (en) 2006-10-12 2008-11-06 Monsanto Technology, Llc Plant micrornas and methods of use thereof
US20090044297A1 (en) * 1999-05-06 2009-02-12 Andersen Scott E Transgenic plants with enhanced agronomic traits
US20090049573A1 (en) * 2002-10-02 2009-02-19 Dotson Stanton B Transgenic plants with enhanced agronomic traits
WO2010002984A1 (en) 2008-07-01 2010-01-07 Monsanto Technology, Llc Recombinant dna constructs and methods for modulating expression of a target gene
WO2010075143A1 (en) 2008-12-22 2010-07-01 Monsanto Technology Llc Genes and uses for plant enhancement
EP2272968A2 (de) 2005-09-08 2011-01-12 Chromatin, Inc. Mit Minichromosomen modifizierte Pflanzen
EP2272967A1 (de) 2003-10-02 2011-01-12 Monsanto Technology LLC Sammeln von Kulturpflanzenverbesserungsmerkmalen bei transgenen Pflanzen
EP2292773A1 (de) 2004-03-25 2011-03-09 Monsanto Technology LLC Gene und ihre Verwendung zur Pflanzenverbesserung
EP2316957A2 (de) 2006-05-12 2011-05-04 Monsanto Technology LLC Verfahren und Zusammensetzungen zur Erzeugung markerfreier transgener Pflanzen
WO2011091311A2 (en) 2010-01-22 2011-07-28 Dow Agrosciences Llc Excision of transgenes in genetically modified organisms
EP2361986A1 (de) 2006-05-16 2011-08-31 Monsanto Technology LLC Verwendung einer nichtagrobakteriellen Spezies zur Pflanzenumwandlung
EP2454940A2 (de) 2006-10-25 2012-05-23 Monsanto Technology LLC Feldfruchtbausysteme zur Unterdrückung von Unkräuten
WO2012092544A2 (en) 2010-12-30 2012-07-05 Dow Agrosciences Llc Nucleic acid molecules that confer resistance to coleopteran pests
EP2478760A1 (de) 2005-05-10 2012-07-25 Monsanto Technology LLC Gene und ihre Verwendung zur Pflanzenverbesserung
EP2489726A2 (de) 2005-01-12 2012-08-22 Monsanto Technology LLC Gene und ihre Verwendung zur Pflanzenverbesserung
WO2013116731A1 (en) 2012-02-02 2013-08-08 Dow Agrosciences Llc Plant transactivation interaction motifs and uses thereof
WO2013116758A1 (en) 2012-02-01 2013-08-08 Dow Agrosciences Llc Synthetic brassica-derived chloroplast transit peptides
WO2013158766A1 (en) 2012-04-17 2013-10-24 Dow Agrosciences Llc Synthetic brassica-derived chloroplast transit peptides
US20130309722A1 (en) * 2011-01-31 2013-11-21 Novozymes A/S Use of browned glucose as a feed substrate
WO2014033723A1 (en) 2012-09-03 2014-03-06 A.B. Seeds Ltd. Method of improving abiotic stress tolerance of plants and plants generated thereby
WO2014039692A2 (en) 2012-09-07 2014-03-13 Dow Agrosciences Llc Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
WO2015038734A2 (en) 2013-09-13 2015-03-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
WO2015095750A1 (en) 2013-12-20 2015-06-25 Dow Agrosciences Llc Rnapii-140 nucleic acid molecules that confer resistance to coleopteran pests
WO2015095774A1 (en) 2013-12-20 2015-06-25 Dow Agrosciences Llc Ras opposite (rop) and related nucleic acid molecules that confer resistance to coleopteran and/or hemipteran pests
US9121022B2 (en) 2010-03-08 2015-09-01 Monsanto Technology Llc Method for controlling herbicide-resistant plants
WO2015171784A1 (en) 2014-05-07 2015-11-12 Dow Agrosciences Llc Dre4 nucleic acid molecules that confer resistance to coleopteran pests
US9290773B2 (en) 2002-10-02 2016-03-22 Monsanto Technology Llc Transgenic plants with enhanced agronomic traits
US9322031B2 (en) 1999-05-06 2016-04-26 Monsanto Technology Llc Transgenic plants with enhanced agronomic traits
WO2016118762A1 (en) 2015-01-22 2016-07-28 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
US9416363B2 (en) 2011-09-13 2016-08-16 Monsanto Technology Llc Methods and compositions for weed control
US9422557B2 (en) 2011-09-13 2016-08-23 Monsanto Technology Llc Methods and compositions for weed control
US9422558B2 (en) 2011-09-13 2016-08-23 Monsanto Technology Llc Methods and compositions for weed control
WO2016144688A1 (en) 2015-03-11 2016-09-15 Pioneer Hi Bred International Inc Insecticidal combinations of pip-72 and methods of use
WO2016196247A1 (en) 2015-05-29 2016-12-08 Dow Agrosciences Llc Spt5 nucleic acid molecules to control insect pests
US9528121B2 (en) 2007-02-20 2016-12-27 Monsanto Technology Llc Invertebrate microRNAs
US9540642B2 (en) 2013-11-04 2017-01-10 The United States Of America, As Represented By The Secretary Of Agriculture Compositions and methods for controlling arthropod parasite and pest infestations
WO2017040343A1 (en) 2015-08-28 2017-03-09 Pioneer Hi-Bred International, Inc. Ochrobactrum-mediated transformation of plants
WO2017059341A1 (en) 2015-10-02 2017-04-06 Monsanto Technology Llc Recombinant maize b chromosome sequence and uses thereof
US9777288B2 (en) 2013-07-19 2017-10-03 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
US9850496B2 (en) 2013-07-19 2017-12-26 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
EP3275308A1 (de) 2010-12-17 2018-01-31 Monsanto Technology LLC Verfahren zur verbesserung der kompetenz von pflanzenzellen
US9976152B2 (en) 2007-06-26 2018-05-22 Monsanto Technology Llc Temporal regulation of gene expression by microRNAs
EP3339440A1 (de) 2010-12-30 2018-06-27 Dow AgroSciences LLC Auf die v-atpase-c-untereinheit gerichtete und resistenz gegenüber coleopteran-schädlingen verleihende nukleinsäuremoleküle
EP3339441A1 (de) 2005-10-13 2018-06-27 Monsanto Technology LLC Verfahren zur herstellung von hybridsaat
EP3342780A1 (de) 2016-12-30 2018-07-04 Dow AgroSciences LLC Nukleinsäuremoleküle mit prä-mrna-verarbeitungsfaktor 8 (prp8) zur kontrolle von insektenschädlingen
EP3354735A1 (de) 2010-12-30 2018-08-01 Dow AgroSciences LLC Auf kleines rho1-gtp-bindendes protein abzielende und resistenz gegenüber coleopteran-schädlingen verleihende nukleinsäuremoleküle
US10041068B2 (en) 2013-01-01 2018-08-07 A. B. Seeds Ltd. Isolated dsRNA molecules and methods of using same for silencing target molecules of interest
EP3375878A1 (de) 2010-12-30 2018-09-19 Dow AgroSciences LLC Auf die v-atpase-h-untereinheit gerichtete und resistenz gegenüber coleopteran-schädlingen verleihende nukleinsäuremoleküle
EP3406715A1 (de) 2012-09-07 2018-11-28 Dow AgroSciences LLC Fad3-performance-loci und zugehörige zielstellenspezifische bindungsproteine zur induzierung zielgerichteter brüche
EP3420809A1 (de) 2014-04-01 2019-01-02 Monsanto Technology LLC Zusammensetzungen und verfahren zur bekämpfung von insektenbefall
US10240162B2 (en) 2012-05-24 2019-03-26 A.B. Seeds Ltd. Compositions and methods for silencing gene expression
US10334848B2 (en) 2014-01-15 2019-07-02 Monsanto Technology Llc Methods and compositions for weed control using EPSPS polynucleotides
US10378012B2 (en) 2014-07-29 2019-08-13 Monsanto Technology Llc Compositions and methods for controlling insect pests
US10557138B2 (en) 2013-12-10 2020-02-11 Beeologics, Inc. Compositions and methods for virus control in Varroa mite and bees
US10568328B2 (en) 2013-03-15 2020-02-25 Monsanto Technology Llc Methods and compositions for weed control
US10612019B2 (en) 2013-03-13 2020-04-07 Monsanto Technology Llc Methods and compositions for weed control
US10609930B2 (en) 2013-03-13 2020-04-07 Monsanto Technology Llc Methods and compositions for weed control
US10655136B2 (en) 2015-06-03 2020-05-19 Monsanto Technology Llc Methods and compositions for introducing nucleic acids into plants
US10683505B2 (en) 2013-01-01 2020-06-16 Monsanto Technology Llc Methods of introducing dsRNA to plant seeds for modulating gene expression
US10760086B2 (en) 2011-09-13 2020-09-01 Monsanto Technology Llc Methods and compositions for weed control
US10801028B2 (en) 2009-10-14 2020-10-13 Beeologics Inc. Compositions for controlling Varroa mites in bees
US10808249B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US10806146B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US10829828B2 (en) 2011-09-13 2020-11-10 Monsanto Technology Llc Methods and compositions for weed control
EP3748003A1 (de) 2016-01-26 2020-12-09 Monsanto Technology LLC Zusammensetzungen und verfahren zur bekämpfung von insektenbefall
US10883103B2 (en) 2015-06-02 2021-01-05 Monsanto Technology Llc Compositions and methods for delivery of a polynucleotide into a plant
US10888579B2 (en) 2007-11-07 2021-01-12 Beeologics Inc. Compositions for conferring tolerance to viral disease in social insects, and the use thereof
US10988764B2 (en) 2014-06-23 2021-04-27 Monsanto Technology Llc Compositions and methods for regulating gene expression via RNA interference
US10995327B2 (en) 2015-12-29 2021-05-04 Monsanto Technology Llc CRISPR-associated transposases and uses thereof
CN113785067A (zh) * 2019-03-04 2021-12-10 塞洛尔克斯股份公司 氯化物诱导型原核表达系统
DE212020000516U1 (de) 2019-03-07 2022-01-17 The Regents of the University of California CRISPR-CAS-Effektorpolypeptide
CN113943689A (zh) * 2021-10-20 2022-01-18 南京鼓楼医院 一种免疫激活性重组乳酸乳球菌的构建及作为肿瘤疫苗和免疫佐剂等方面的应用
WO2023009547A1 (en) 2021-07-26 2023-02-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and uses thereof
US11578334B2 (en) 2017-10-25 2023-02-14 Monsanto Technology Llc Targeted endonuclease activity of the RNA-guided endonuclease CasX in eukaryotes
US11692182B2 (en) 2015-10-09 2023-07-04 Monsanto Technology Llc RNA-guided DNA nucleases and uses thereof
US11732268B2 (en) 2016-06-28 2023-08-22 Monsanto Technology Llc Methods and compositions for use in genome modification in plants
US11807857B2 (en) 2014-06-25 2023-11-07 Monsanto Technology Llc Methods and compositions for delivering nucleic acids to plant cells and regulating gene expression
US11920140B2 (en) 2017-08-22 2024-03-05 Napigen, Inc. Organelle genome modification using polynucleotide guided endonuclease
US11981900B2 (en) 2021-09-09 2024-05-14 Monsanto Technology Llc Increasing gene editing and site-directed integration events utilizing meiotic and germline promoters

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3772226B1 (de) 2019-07-30 2023-01-25 Volkswagen AG Verfahren, computerprogramme und vorrichtungen für ein befehlszentrum und fahrzeug

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031562A1 (en) * 1994-05-12 1995-11-23 Quest International B.V. Process for inhibiting the growth of a culture of lactic acid bacteria, and optionally lysing the bacterial cells, and uses of the resulting lysed culture
WO1995031563A1 (en) * 1994-05-12 1995-11-23 Quest International B.V. Complex inducible promoter system derivable from a phage of a lactic acid bacterium (lab), and its use in a lab for production of a desired protein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031562A1 (en) * 1994-05-12 1995-11-23 Quest International B.V. Process for inhibiting the growth of a culture of lactic acid bacteria, and optionally lysing the bacterial cells, and uses of the resulting lysed culture
WO1995031563A1 (en) * 1994-05-12 1995-11-23 Quest International B.V. Complex inducible promoter system derivable from a phage of a lactic acid bacterium (lab), and its use in a lab for production of a desired protein

Cited By (158)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030027358A1 (en) * 1997-04-24 2003-02-06 Immunex Corporation Family of immunoregulators designated leukocyte immunoglobulin-like receptors (LIR)
US9322031B2 (en) 1999-05-06 2016-04-26 Monsanto Technology Llc Transgenic plants with enhanced agronomic traits
US20090044297A1 (en) * 1999-05-06 2009-02-12 Andersen Scott E Transgenic plants with enhanced agronomic traits
US20090241217A9 (en) * 2001-05-22 2009-09-24 Jingrui Wu Yield-improved transgenic plants
US20080104730A1 (en) * 2001-05-22 2008-05-01 Jingrui Wu Yield-improved transgenic plants
US20060078564A1 (en) * 2002-05-08 2006-04-13 Immunex Corporation Family of immunoregulators designated leukocyte immunoglobulin-like receptors (LIR)
US9290773B2 (en) 2002-10-02 2016-03-22 Monsanto Technology Llc Transgenic plants with enhanced agronomic traits
US20090049573A1 (en) * 2002-10-02 2009-02-19 Dotson Stanton B Transgenic plants with enhanced agronomic traits
EP1881074A2 (de) 2003-05-05 2008-01-23 Monsanto Technology, LLC Transgene Pflanzen mit erhöhtem Glycin-Betain
US20050281914A1 (en) * 2003-06-20 2005-12-22 Steele James L Methods and compositions involving endopeptidases PepO2 and PepO3
US7741438B2 (en) 2003-06-20 2010-06-22 Wisconsin Alumni Research Foundation Methods and compositions involving endopeptidases PepO2 and PepO3
EP2272967A1 (de) 2003-10-02 2011-01-12 Monsanto Technology LLC Sammeln von Kulturpflanzenverbesserungsmerkmalen bei transgenen Pflanzen
EP3290516A1 (de) 2004-02-10 2018-03-07 Monsanto Technology LLC Rekombinante dna zur genunterdrückung
EP2365072A1 (de) 2004-02-10 2011-09-14 Monsanto Technology LLC Rekombinante DNA zur Genunterdrückung
WO2005077116A2 (en) 2004-02-10 2005-08-25 Mosanto Technology, Llc Recombinant dna for gene suppression
EP2292773A1 (de) 2004-03-25 2011-03-09 Monsanto Technology LLC Gene und ihre Verwendung zur Pflanzenverbesserung
EP2295582A2 (de) 2004-07-31 2011-03-16 Monsanto Technology LLC Gene und ihre Verwendung zur Pflanzenverbesserung
EP1621632A1 (de) 2004-07-31 2006-02-01 Monsanto Technology, LLC Gene und ihre Verwendung zur Pflanzenverbesserung
EP3372676A1 (de) 2004-12-21 2018-09-12 Monsanto Technology, LLC Rekombinante dna-konstrukte und verfahren zur steuerung der gen-expression
WO2006073727A2 (en) 2004-12-21 2006-07-13 Monsanto Technology, Llc Recombinant dna constructs and methods for controlling gene expression
US10793869B2 (en) 2004-12-21 2020-10-06 Monsanto Technology Llc Recombinant DNA constructs and methods for controlling gene expression
US9708620B2 (en) 2004-12-21 2017-07-18 Monsanto Technology Llc Recombinant DNA constructs and methods for controlling gene expression
EP2765189A1 (de) 2004-12-21 2014-08-13 Monsanto Technology LLC Rekombinante DNA-Konstrukte und Verfahren zur Steuerung der Gen-Expression
US9000264B2 (en) 2004-12-21 2015-04-07 Monsanto Technology Llc Recombinant DNA constructs and methods for controlling gene expression
US9212370B2 (en) 2004-12-21 2015-12-15 Monsanto Technology Llc Recombinant DNA constructs and methods for controlling gene expression
US20110035839A1 (en) * 2004-12-21 2011-02-10 Linda Lutfiyya Recombinant DNA Constructs and Methods for Controlling Gene Expression
EP2489726A2 (de) 2005-01-12 2012-08-22 Monsanto Technology LLC Gene und ihre Verwendung zur Pflanzenverbesserung
EP2584033A1 (de) 2005-01-12 2013-04-24 Monsanto Technology LLC Gene und ihre Verwendung zur Pflanzenverbesserung
EP3059306A1 (de) 2005-01-12 2016-08-24 Monsanto Technology LLC Gene und verwendungen zur pflanzenverbesserung
EP2478760A1 (de) 2005-05-10 2012-07-25 Monsanto Technology LLC Gene und ihre Verwendung zur Pflanzenverbesserung
WO2007011479A2 (en) 2005-07-19 2007-01-25 Monsanto Technology, Llc Double-stranded rna stabilized in planta
EP2272968A2 (de) 2005-09-08 2011-01-12 Chromatin, Inc. Mit Minichromosomen modifizierte Pflanzen
US9121028B2 (en) 2005-09-09 2015-09-01 Monsanto Technology Llc Selective gene expression in plants
US20070061922A1 (en) * 2005-09-09 2007-03-15 Edgerton Michael D Selective gene expression in plants
WO2007041419A1 (en) 2005-10-03 2007-04-12 Monsanto Technology Llc Transgenic plant seed with increased lysine
EP3339441A1 (de) 2005-10-13 2018-06-27 Monsanto Technology LLC Verfahren zur herstellung von hybridsaat
EP2316958A2 (de) 2006-05-12 2011-05-04 Monsanto Technology LLC Verfahren und Zusammensetzungen zur Erzeugung markerfreier transgener Pflanzen
EP2811025A2 (de) 2006-05-12 2014-12-10 Monsanto Technology LLC Verfahren und Zusammensetzungen zum Erhalt von markerfreien Pflanzen
EP2316957A2 (de) 2006-05-12 2011-05-04 Monsanto Technology LLC Verfahren und Zusammensetzungen zur Erzeugung markerfreier transgener Pflanzen
EP2803728A1 (de) 2006-05-16 2014-11-19 Monsanto Technology LLC Verwendung einer bakteriellen nichtagrobakteriellen Spezies zur Pflanzenumwandlung
EP2361986A1 (de) 2006-05-16 2011-08-31 Monsanto Technology LLC Verwendung einer nichtagrobakteriellen Spezies zur Pflanzenumwandlung
EP3196311A1 (de) 2006-05-16 2017-07-26 Monsanto Technology LLC Verwendung einer nichtagrobakteriellen spezies zur pflanzenumwandlung
EP2371964A1 (de) 2006-05-16 2011-10-05 Monsanto Technology LLC Verwendung einer nichtagrobakteriellen Spezies zur Pflanzenumwandlung
EP3608413A1 (de) 2006-05-16 2020-02-12 Monsanto Technology LLC Verwendung einer nichtagrobakteriellen spezies zur pflanzenumwandlung
WO2007143690A2 (en) 2006-06-06 2007-12-13 Monsanto Technology Llc Methods for weed control
US20080040973A1 (en) * 2006-06-23 2008-02-21 Donald Nelson Transgenic crop plants with improved stress tolerance
EP3378953A1 (de) 2006-10-12 2018-09-26 Monsanto Technology LLC Pflanzen-mikro-rnas und verfahren zur verwendung davon
EP2985353A1 (de) 2006-10-12 2016-02-17 Monsanto Technology LLC Pflanzen-mikro-rnas und verfahren zur verwendung davon
EP2559767A2 (de) 2006-10-12 2013-02-20 Monsanto Technology LLC Pflanzliche Mikro-RNAs und Verfahren zur Verwendung davon
US10435686B2 (en) 2006-10-12 2019-10-08 Monsanto Technology Llc Plant microRNAs and methods of use thereof
US20090070898A1 (en) * 2006-10-12 2009-03-12 Edwards Allen Plant microRNAs and methods of use thereof
WO2008133643A2 (en) 2006-10-12 2008-11-06 Monsanto Technology, Llc Plant micrornas and methods of use thereof
US8946511B2 (en) 2006-10-12 2015-02-03 Monsanto Technology Llc Plant microRNAs and methods of use thereof
WO2008048964A2 (en) 2006-10-16 2008-04-24 Monsanto Technology Llc Methods and compositions for improving plant health
EP2454940A2 (de) 2006-10-25 2012-05-23 Monsanto Technology LLC Feldfruchtbausysteme zur Unterdrückung von Unkräuten
US10570414B2 (en) 2007-02-20 2020-02-25 Monsanto Technology Llc Invertebrate microRNAs
US9528121B2 (en) 2007-02-20 2016-12-27 Monsanto Technology Llc Invertebrate microRNAs
EP2527452A2 (de) 2007-02-26 2012-11-28 Monsanto Technology LLC Chloroplasten-Transitpeptide zun wirksamen Abzielen auf DMO und Verwendungen davon
EP2527453A2 (de) 2007-02-26 2012-11-28 Monsanto Technology LLC Chloroplasten-Transitpeptide zun wirksamen Abzielen auf DMO und Verwendungen davon
WO2008105890A2 (en) 2007-02-26 2008-09-04 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of dmo and uses thereof
WO2008112633A2 (en) 2007-03-09 2008-09-18 Monsanto Technology Llc Method of meristem excision and transformation
WO2008112645A2 (en) 2007-03-09 2008-09-18 Monsanto Technology Llc Methods for plant transformation using spectinomycin selection
EP2811026A2 (de) 2007-03-09 2014-12-10 Monsanto Technology LLC Verfahren zur Pflanzentransformation mittels Spectinomycin-Selektion
EP2450448A1 (de) 2007-03-09 2012-05-09 Monsanto Technology LLC Verfahren zur Pflanzentransformation mittels Spectinomycin-Selektion
US9976152B2 (en) 2007-06-26 2018-05-22 Monsanto Technology Llc Temporal regulation of gene expression by microRNAs
US11008580B2 (en) 2007-06-26 2021-05-18 Monsanto Technology Llc Regulation of gene expression by temporal or leaf specific promoters
US10888579B2 (en) 2007-11-07 2021-01-12 Beeologics Inc. Compositions for conferring tolerance to viral disease in social insects, and the use thereof
EP2924118A1 (de) 2008-07-01 2015-09-30 Monsanto Technology LLC Rekombinante DNA-Konstrukte und Verfahren zur Modulierung der Expression eines Zielgens
WO2010002984A1 (en) 2008-07-01 2010-01-07 Monsanto Technology, Llc Recombinant dna constructs and methods for modulating expression of a target gene
US9040774B2 (en) 2008-07-01 2015-05-26 Monsanto Technology Llc Recombinant DNA constructs encoding ribonuclease cleavage blockers and methods for modulating expression of a target gene
WO2010075143A1 (en) 2008-12-22 2010-07-01 Monsanto Technology Llc Genes and uses for plant enhancement
US10801028B2 (en) 2009-10-14 2020-10-13 Beeologics Inc. Compositions for controlling Varroa mites in bees
WO2011091311A2 (en) 2010-01-22 2011-07-28 Dow Agrosciences Llc Excision of transgenes in genetically modified organisms
US11812738B2 (en) 2010-03-08 2023-11-14 Monsanto Technology Llc Polynucleotide molecules for gene regulation in plants
US9121022B2 (en) 2010-03-08 2015-09-01 Monsanto Technology Llc Method for controlling herbicide-resistant plants
US9988634B2 (en) 2010-03-08 2018-06-05 Monsanto Technology Llc Polynucleotide molecules for gene regulation in plants
EP3275308A1 (de) 2010-12-17 2018-01-31 Monsanto Technology LLC Verfahren zur verbesserung der kompetenz von pflanzenzellen
EP4059342A1 (de) 2010-12-17 2022-09-21 Monsanto Technology LLC Verfahren zur verbesserung der kompetenz von pflanzenzellen
EP3354735A1 (de) 2010-12-30 2018-08-01 Dow AgroSciences LLC Auf kleines rho1-gtp-bindendes protein abzielende und resistenz gegenüber coleopteran-schädlingen verleihende nukleinsäuremoleküle
EP3339440A1 (de) 2010-12-30 2018-06-27 Dow AgroSciences LLC Auf die v-atpase-c-untereinheit gerichtete und resistenz gegenüber coleopteran-schädlingen verleihende nukleinsäuremoleküle
WO2012092544A2 (en) 2010-12-30 2012-07-05 Dow Agrosciences Llc Nucleic acid molecules that confer resistance to coleopteran pests
EP3375878A1 (de) 2010-12-30 2018-09-19 Dow AgroSciences LLC Auf die v-atpase-h-untereinheit gerichtete und resistenz gegenüber coleopteran-schädlingen verleihende nukleinsäuremoleküle
US20130309722A1 (en) * 2011-01-31 2013-11-21 Novozymes A/S Use of browned glucose as a feed substrate
US9347080B2 (en) * 2011-01-31 2016-05-24 Novozymes A/S Use of browned glucose as a feed substrate
US9422558B2 (en) 2011-09-13 2016-08-23 Monsanto Technology Llc Methods and compositions for weed control
US9422557B2 (en) 2011-09-13 2016-08-23 Monsanto Technology Llc Methods and compositions for weed control
US10806146B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US9416363B2 (en) 2011-09-13 2016-08-16 Monsanto Technology Llc Methods and compositions for weed control
US10808249B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US10760086B2 (en) 2011-09-13 2020-09-01 Monsanto Technology Llc Methods and compositions for weed control
US10829828B2 (en) 2011-09-13 2020-11-10 Monsanto Technology Llc Methods and compositions for weed control
WO2013116758A1 (en) 2012-02-01 2013-08-08 Dow Agrosciences Llc Synthetic brassica-derived chloroplast transit peptides
WO2013116731A1 (en) 2012-02-02 2013-08-08 Dow Agrosciences Llc Plant transactivation interaction motifs and uses thereof
WO2013158766A1 (en) 2012-04-17 2013-10-24 Dow Agrosciences Llc Synthetic brassica-derived chloroplast transit peptides
US10240161B2 (en) 2012-05-24 2019-03-26 A.B. Seeds Ltd. Compositions and methods for silencing gene expression
US10934555B2 (en) 2012-05-24 2021-03-02 Monsanto Technology Llc Compositions and methods for silencing gene expression
US10240162B2 (en) 2012-05-24 2019-03-26 A.B. Seeds Ltd. Compositions and methods for silencing gene expression
WO2014033723A1 (en) 2012-09-03 2014-03-06 A.B. Seeds Ltd. Method of improving abiotic stress tolerance of plants and plants generated thereby
EP3431600A1 (de) 2012-09-07 2019-01-23 Dow AgroSciences LLC Fad2-leistungsorte und entsprechende zielstellenspezifische bindungsproteine zur herbeiführung zielgerichteter brüche
WO2014039692A2 (en) 2012-09-07 2014-03-13 Dow Agrosciences Llc Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
EP3404099A1 (de) 2012-09-07 2018-11-21 Dow AgroSciences LLC Fad2-leistungsorte und entsprechende zielstellenspezifische bindungsproteine zur herbeiführung zielgerichteter brüche
EP3406715A1 (de) 2012-09-07 2018-11-28 Dow AgroSciences LLC Fad3-performance-loci und zugehörige zielstellenspezifische bindungsproteine zur induzierung zielgerichteter brüche
WO2014039702A2 (en) 2012-09-07 2014-03-13 Dow Agrosciences Llc Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10683505B2 (en) 2013-01-01 2020-06-16 Monsanto Technology Llc Methods of introducing dsRNA to plant seeds for modulating gene expression
US10041068B2 (en) 2013-01-01 2018-08-07 A. B. Seeds Ltd. Isolated dsRNA molecules and methods of using same for silencing target molecules of interest
US10609930B2 (en) 2013-03-13 2020-04-07 Monsanto Technology Llc Methods and compositions for weed control
US10612019B2 (en) 2013-03-13 2020-04-07 Monsanto Technology Llc Methods and compositions for weed control
US10568328B2 (en) 2013-03-15 2020-02-25 Monsanto Technology Llc Methods and compositions for weed control
US9850496B2 (en) 2013-07-19 2017-12-26 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US11377667B2 (en) 2013-07-19 2022-07-05 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US9777288B2 (en) 2013-07-19 2017-10-03 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
EP3608412A2 (de) 2013-07-19 2020-02-12 Monsanto Technology LLC Zusammensetzungen und verfahren zur bekämpfung von leptinotarsa
US9856495B2 (en) 2013-07-19 2018-01-02 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US10597676B2 (en) 2013-07-19 2020-03-24 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
EP4159028A1 (de) 2013-09-13 2023-04-05 Pioneer Hi-Bred International, Inc. Insektizidproteine und verfahren zu deren verwendung
EP3692786A1 (de) 2013-09-13 2020-08-12 Pioneer Hi-Bred International, Inc. Insektizide proteine und verfahren zu deren verwendung
WO2015038734A2 (en) 2013-09-13 2015-03-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
US9540642B2 (en) 2013-11-04 2017-01-10 The United States Of America, As Represented By The Secretary Of Agriculture Compositions and methods for controlling arthropod parasite and pest infestations
US10100306B2 (en) 2013-11-04 2018-10-16 Monsanto Technology Llc Compositions and methods for controlling arthropod parasite and pest infestations
US10927374B2 (en) 2013-11-04 2021-02-23 Monsanto Technology Llc Compositions and methods for controlling arthropod parasite and pest infestations
US10557138B2 (en) 2013-12-10 2020-02-11 Beeologics, Inc. Compositions and methods for virus control in Varroa mite and bees
WO2015095750A1 (en) 2013-12-20 2015-06-25 Dow Agrosciences Llc Rnapii-140 nucleic acid molecules that confer resistance to coleopteran pests
WO2015095774A1 (en) 2013-12-20 2015-06-25 Dow Agrosciences Llc Ras opposite (rop) and related nucleic acid molecules that confer resistance to coleopteran and/or hemipteran pests
US10334848B2 (en) 2014-01-15 2019-07-02 Monsanto Technology Llc Methods and compositions for weed control using EPSPS polynucleotides
US11091770B2 (en) 2014-04-01 2021-08-17 Monsanto Technology Llc Compositions and methods for controlling insect pests
EP3420809A1 (de) 2014-04-01 2019-01-02 Monsanto Technology LLC Zusammensetzungen und verfahren zur bekämpfung von insektenbefall
WO2015171784A1 (en) 2014-05-07 2015-11-12 Dow Agrosciences Llc Dre4 nucleic acid molecules that confer resistance to coleopteran pests
US10988764B2 (en) 2014-06-23 2021-04-27 Monsanto Technology Llc Compositions and methods for regulating gene expression via RNA interference
US11807857B2 (en) 2014-06-25 2023-11-07 Monsanto Technology Llc Methods and compositions for delivering nucleic acids to plant cells and regulating gene expression
US11124792B2 (en) 2014-07-29 2021-09-21 Monsanto Technology Llc Compositions and methods for controlling insect pests
US10378012B2 (en) 2014-07-29 2019-08-13 Monsanto Technology Llc Compositions and methods for controlling insect pests
US10968449B2 (en) 2015-01-22 2021-04-06 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
WO2016118762A1 (en) 2015-01-22 2016-07-28 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
WO2016144688A1 (en) 2015-03-11 2016-09-15 Pioneer Hi Bred International Inc Insecticidal combinations of pip-72 and methods of use
WO2016196247A1 (en) 2015-05-29 2016-12-08 Dow Agrosciences Llc Spt5 nucleic acid molecules to control insect pests
US10883103B2 (en) 2015-06-02 2021-01-05 Monsanto Technology Llc Compositions and methods for delivery of a polynucleotide into a plant
US10655136B2 (en) 2015-06-03 2020-05-19 Monsanto Technology Llc Methods and compositions for introducing nucleic acids into plants
WO2017040343A1 (en) 2015-08-28 2017-03-09 Pioneer Hi-Bred International, Inc. Ochrobactrum-mediated transformation of plants
US11732269B2 (en) 2015-10-02 2023-08-22 Monsanto Technology Llc Recombinant maize B chromosome sequence and uses thereof
WO2017059341A1 (en) 2015-10-02 2017-04-06 Monsanto Technology Llc Recombinant maize b chromosome sequence and uses thereof
US11692182B2 (en) 2015-10-09 2023-07-04 Monsanto Technology Llc RNA-guided DNA nucleases and uses thereof
US10995327B2 (en) 2015-12-29 2021-05-04 Monsanto Technology Llc CRISPR-associated transposases and uses thereof
EP4159848A1 (de) 2015-12-29 2023-04-05 Monsanto Technology LLC Neuartige crispr-assoziierte transposasen und verwendungen davon
EP4159849A1 (de) 2015-12-29 2023-04-05 Monsanto Technology LLC Neuartige crispr-assoziierte transposasen und verwendungen davon
EP4159847A1 (de) 2015-12-29 2023-04-05 Monsanto Technology LLC Neuartige crispr-assoziierte transposasen und verwendungen davon
EP3748003A1 (de) 2016-01-26 2020-12-09 Monsanto Technology LLC Zusammensetzungen und verfahren zur bekämpfung von insektenbefall
US11732268B2 (en) 2016-06-28 2023-08-22 Monsanto Technology Llc Methods and compositions for use in genome modification in plants
EP3342780A1 (de) 2016-12-30 2018-07-04 Dow AgroSciences LLC Nukleinsäuremoleküle mit prä-mrna-verarbeitungsfaktor 8 (prp8) zur kontrolle von insektenschädlingen
US11920140B2 (en) 2017-08-22 2024-03-05 Napigen, Inc. Organelle genome modification using polynucleotide guided endonuclease
US11578334B2 (en) 2017-10-25 2023-02-14 Monsanto Technology Llc Targeted endonuclease activity of the RNA-guided endonuclease CasX in eukaryotes
CN113785067A (zh) * 2019-03-04 2021-12-10 塞洛尔克斯股份公司 氯化物诱导型原核表达系统
CN113785067B (zh) * 2019-03-04 2024-01-16 奥瑞丽斯治疗股份公司 氯化物诱导型原核表达系统
EP4219700A1 (de) 2019-03-07 2023-08-02 The Regents of the University of California Crispr-cas-effektorpolypeptide und verfahren zur verwendung davon
DE212020000516U1 (de) 2019-03-07 2022-01-17 The Regents of the University of California CRISPR-CAS-Effektorpolypeptide
WO2023009547A1 (en) 2021-07-26 2023-02-02 Flagship Pioneering Innovations Vi, Llc Trem compositions and uses thereof
US11981900B2 (en) 2021-09-09 2024-05-14 Monsanto Technology Llc Increasing gene editing and site-directed integration events utilizing meiotic and germline promoters
CN113943689A (zh) * 2021-10-20 2022-01-18 南京鼓楼医院 一种免疫激活性重组乳酸乳球菌的构建及作为肿瘤疫苗和免疫佐剂等方面的应用
CN113943689B (zh) * 2021-10-20 2024-02-09 南京鼓楼医院 一种免疫激活性重组乳酸乳球菌的构建及作为肿瘤疫苗和免疫佐剂等方面的应用

Also Published As

Publication number Publication date
EP0865496A1 (de) 1998-09-23
WO1998010080A1 (en) 1998-03-12
ID18254A (id) 1998-03-19
JPH11514534A (ja) 1999-12-14
AU4208797A (en) 1998-03-26
CA2236770A1 (en) 1998-03-12

Similar Documents

Publication Publication Date Title
US6140078A (en) Salt-inducible promoter derivable from a lactic acid bacterium, and its use in a lactic acid bacterium for production of a desired protein
van Belkum et al. Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6
Diep et al. A bacteriocin‐like peptide induces bacteriocin synthesis in Lactobacillus plantarum C11
van Belkum et al. Organization and nucleotide sequences of two lactococcal bacteriocin operons
Emond et al. Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis
Geller et al. Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2
NZ525394A (en) Improved fermentation method for production of heterologous gene products in lactic acid bacteria
Bouchard et al. Characterization of the two-component abortive phage infection mechanism AbiT from Lactococcus lactis
US6030807A (en) Highly regulable promoter for heterologous gene expression
Vesanto et al. Characterization and expression of the Lactobacillus helveticus pepC gene encoding a general aminopeptidase
Kok Inducible gene expression and environmentally regulated genes in lactic acid bacteria
US6133023A (en) Lactic acid bacterial regulatable expression system
Mayo et al. Cloning and expression of the plasmid encoded β-D-galactosidase gene from a Lactobacillus plantarum strain of dairy origin
WO1998011231A9 (en) Highly regulable promoter for heterologous gene expression
Duan et al. Identification and characterization of a mobilizing plasmid, pND300, in Lactococcus lactis M189 and its encoded nisin resistance determinant
Kim et al. Bacteriophage resistance in Lactococcus lactis ssp. lactis using antisense ribonucleic acid
AU6277490A (en) Promoter probe vectors, able to replicate in e.coli, b.subtilis, lactococci and lactobacillus as well as uses thereof
ES2266871T3 (es) Casetes de expresion procariotas reguladas por el cinc.
US5939317A (en) Use of a Sec-dependent secretion system for secreting proteins that are usually secreted by a Sec-independent secretion system, bacteria containing it and their use
WO1994000581A1 (en) Lactobacillus expression system using surface protein gene sequences
US5994118A (en) DNA encoding phage resistance protein
AU769084B2 (en) The lactose operon of lactobacillus delbrueckii and its use for controlling gene transcription and/or expression in bacterial cells
US7119167B2 (en) Fragment of L. lactis plasmid conferring antiphage activity and uses thereof
AU8081691A (en) Novel bacteriocin from a strain of lactococcus lactis subsp. cremoris
Yu et al. Restriction mapping and localization of the lactose-metabolizing genes of Streptococcus cremoris pDI-21

Legal Events

Date Code Title Description
AS Assignment

Owner name: UNILEVER PATENT HOLDINGS, NETHERLANDS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SANDERS, JAN WILLEM;VENEMA, GERARD;LEDEBOER, ADRIANUS MARINUS;REEL/FRAME:009394/0063;SIGNING DATES FROM 19980604 TO 19980616

FEPP Fee payment procedure

Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

REMI Maintenance fee reminder mailed
LAPS Lapse for failure to pay maintenance fees
STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362

FP Lapsed due to failure to pay maintenance fee

Effective date: 20041031